Unassisted Establishment of Biological Soil Crusts on Dryland Road Slopes

Web Ecol., 19, 39–51, 2019 https://doi.org/10.5194/we-19-39-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License.

Unassisted establishment of biological soil crusts on dryland road slopes

Laura Concostrina-Zubiri, Juan M. Arenas, Isabel Martínez, and Adrián Escudero Área de Biodiversidad y Conservación, Departamento de Biología, Geología, Física y Química Inorgánica, Universidad Rey Juan Carlos, Móstoles, 28933 Madrid, Spain Correspondence: Laura Concostrina-Zubiri ([email protected]) and Juan M. Arenas ([email protected])

Received: 13 January 2019 – Revised: 12 May 2019 – Accepted: 15 May 2019 – Published: 6 June 2019

Abstract. Understanding patterns of habitat natural recovery after human-made disturbances is critical for the conservation of ecosystems under high environmental stress, such as drylands. In particular, the unassisted establishment of nonvascular plants such as biological soil crusts or biocrust communities (e.g., soil lichens, mosses and cyanobacteria) in newly formed habitats is not yet fully understood. However, the potential of biocrusts to improve soil structure and function at the early stages of succession and promote ecosystem recovery is enormous. In this study, we evaluated the capacity of lichen biocrusts to spontaneously establish and develop on road slopes in a Mediterranean shrubland. We also compared taxonomic and functional diversity of biocrusts between road slopes and natural habitats in the surroundings. Biocrust richness and cover, species composition, and functional structure were measured in 17 road slopes (nine roadcuts and eight embankments) along a 13 km highway stretch. Topography, soil properties and vascular plant communities of road slopes were also characterized. We used Kruskal–Wallis tests and applied redundancy analysis (RDA) to test the effect of environmental scenario (road slopes vs. natural habitat) and other local factors on biocrust features. We found that biocrusts were common in road slopes after ∼ 20 years of construction with no human assistance needed. However, species richness and cover were still lower than in natural remnants. Also, functional structure was quite similar between roadcuts (i.e., after soil excavation) and natural remnants, and topography and soil properties influenced species composition while environmental scenario type and vascular plant cover did not. These findings further support the idea of biocrusts as promising restoration tools in drylands and confirm the critical role of edaphic factors in biocrust establishment and development in land-use change scenarios.

1 Introduction (Delgado-Baquerizo et al., 2015) and hydrology (Chamizo et al., 2013), and also modulating biotic interactions (Luzuriaga Biological soil crusts (biocrusts) have earned a place as key et al., 2012). Moreover, due to the functional roles they per- ecosystem components in drylands in the last decades (We- form and their ability to establish and develop in very harsh ber et al., 2016a). They are complex communities composed environments, biocrusts have been pointed out as promis- of lichens, bryophytes, fungi, cyanobacteria and other mi- ing tools in dryland restoration (Bowker, 2007). Some of the croorganisms living on and interacting with the ﬁrst soil biocrust constituents are early, fast colonizers of degraded ar- layers, and are found in almost every dryland occurring in eas with enormous potential to aid and speed up ecosystem stressful conditions, i.e., water scarcity and high solar radia- structure and functioning recovery, e.g., stabilizing soil sur- tion and temperatures. Biocrusts contribute greatly to ecosys- face, improving soil fertility, maintaining soil humidity and tem diversity, with several tens of species per square me- promoting the establishment of vascular plants (Weber et al., tre, and to ecosystem functioning, being critical in the ﬁx- 2016a). This is particularly true in strongly disturbed areas ation of carbon and nitrogen (Elbert et al., 2012), controlling under high abiotic stress, where vascular plant development topsoil structure (Jimenez Aguilar et al., 2009), chemistry

Published by Copernicus Publications on behalf of the European Ecological Federation (EEF). 40 L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes is less likely to occur due to a higher demand on soil re- sitional level (Arenas et al., 2017a). For biocrusts, we ex- sources and lower tolerance of water scarcity, among others pect to find processes of colonization and community as- (Bowker, 2007). sembly on road slopes similar to those that occur for vas- Biocrusts have been shown to recover naturally without cular vegetation, and therefore evidence the ecological in- assistance from mechanical disturbances, such as grazing, terest of natural colonization as a useful biocrust restora- trampling and vehicle driving, and also from fire, at varying tion measure. In particular, we hypothesized that biocrusts recovery rates and following different community trajecto- establish easily in the road slopes, provided that biocrusts ries depending on the disturbance type, intensity and timing are common and abundant in the surroundings (i.e., inocu- (Belnap and Eldridge, 2003). However, little is known about lum availability) and because of their capacity to tolerate natural recovery of biocrusts in newly formed and emer- harsh environmental conditions typical for newly created gent habitats such as those generated after the construction habitats (e.g., very thin soil, high radiation inputs) (Weber of linear infrastructures which include massive soil removal et al. ,2016b). Nevertheless, we expect to find marked differ- or the accumulation of exogenous soil material. At the very ences in biocrust composition depending on road slope type fine spatial scale (i.e., topsoil, area < 10 m2), several studies (i.e., roadcut vs. embankment), topography, soil properties have evaluated biocrust natural colonization and establish- and vascular plant abundance since the diversity of these or- ment after biocrust removal (i.e., scalping). Recovery rates ganisms is highly sensitive to changes in (i) climate at the range from less than 3 years for cyanobacteria, commonly local and microscale given by topography, e.g., sun vs. shade the earliest, fastest colonizers within biocrusts, up to a few environments (Pintado et al., 2005); (ii) soil physicochemical decades or almost a millennium for bryophytes and lichens, properties, e.g., texture (Fischer and Subbotina, 2014), nutri- the typical components of more developed, mature biocrusts ent content (Bowker et al., 2006) and pH (Ochoa-Hueso et (reviewed in Weber et al., 2016b). Similarly to biocrust re- al., 2011); and (iii) biotic interactions, especially with vascu- covery in secondary succession, the colonization and estab- lar plants, e.g., biocrust–plant facilitation–competition pro- lishment of biocrusts largely depend on climate, particularly, cesses (reviewed in Bowker et al., 2016). In this study, we precipitation amount and timing after disturbance, and soil aimed to assess the potential of lichen biocrusts to colonize properties, such as soil stability and nutrient concentration and prosper in pioneer habitats under natural conditions (i.e., (Weber et al., 2016b). with no artificial assistance). We focused only on lichens be- Surprisingly, to our knowledge, there is only one study cause they are the most conspicuous, rich and common com- focusing entirely on the recovery of biocrust communities ponents of biocrusts in Mediterranean grasslands and shrub- on road slopes (Chiquoine et al., 2016). Yet, Chiquoine lands (Concostrina-Zubiri et al., 2014), even in fragmented et al. (2016) did not focus on natural colonization but on landscapes (Concostrina-Zubiri et al., 2018). successful establishment after applying biocrust inoculum, Specifically, we addressed the following questions: (1) can among other restoration treatments. This is a bit striking lichen biocrusts establish on road slopes spontaneously? If since there is no baseline for comparison. Therefore, no so, (2) what species and functional attributes characterize previous study has evaluated the unassisted recovery of the newly formed communities? Regarding the functional biocrusts on road slopes, and this limited knowledge on attributes characterizing these communities, we focused on biocrust recovery under natural conditions weakens many eutrophication tolerance, gypsum tolerance, because our projects of ecological restoration of road slopes in drylands. biocrust model occurs in this type of soil (Concostrina-Zubiri In contrast, many studies have highlighted the role of nat- et al., 2018), and thallus continuity to test the following hy- ural colonization in the establishment of vascular vegeta- potheses: (i) lichen biocrust communities in the road slopes tion on road slopes (Bochet et al., 2007; Mola et al., 2011; are more tolerant to high nutrient levels (e.g., derived from Arenas et al., 2017a, b). In Mediterranean drylands, na- the road traffic and the subsequent passive air fertilization), tive vascular plants can establish and develop well in road (ii) they present a higher tolerance of/affinity to gypsum con- slopes in the absence of human assistance when the slope is tent, as expected in a less developed topsoil layer at road gentle (< 45◦) (Bochet and García-Fayos, 2004) and suffi- slopes compared to natural remnants where soil carbon and cient propagule is available from the surroundings (Arenas other nutrients had higher levels (Arenas et al., 2017a); and et al., 2017b). However, species composition differs notably (iii) lichen biocrusts with a less continuous thallus are more from communities found in the natural surrounding areas, likely to colonize and prosper in road slopes, characterized mainly due to topography and modification of initial soil by a more hostile microenvironment. (3) What is the role conditions derived from the process of road slope construc- of habitat scenario type (e.g., road slopes vs. natural areas) tion (Bochet and García-Fayos, 2004; Arenas et al., 2017a). and other local factors (i.e., topography, soil properties and Moreover, within road slopes, roadcuts and embankments vascular plant communities) in determining the structure and have very different structural properties (e.g., soil removal composition of lichen biocrusts? By addressing these ques- vs. soil application, respectively) and environmental char- tions, we could identify the factors that determine the natural acteristics, and as a result, further differences can be found colonization of biocrust in road slopes, and point out some between their respective plant communities at the compo- restoration measures to carry out in road slopes for promot-

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their structural and physicochemical properties; (i) roadcuts (n = 9), which are constructed by excavation, resulting in areas of bare soil and exposed bedrock; and (ii) embankments (n = 8), which are constructed by heaping and compacting materials and eventually receiving topsoil treatments (Tormo et al., 2009). At each road slope, we established a central sampling plot (2.40 m × 2.40 m) and regularly divided it in 64 quadrats (30 cm × 30 cm cells). Then, we randomly selected 10 quadrats to estimate lichen biocrust species abundance visually as the percentage cover per species. We also sampled lichen biocrust communities in natural habitat remnants adjacent to the highway to compare lichen biocrust communities colonizing the emergent habitats (road slopes) to natural, well-established lichen biocrusts. To do so, we selected 50 natural shrubland remnants immersed in an agri- cultural matrix in a ca. 76 km2 area comprising two ad- Figure 1. Map of the study area showing the road (in black), jacent 3 km rectangles at both sides of the 13 km stretch roadcuts (squares), embankments (circles) and natural remnants (in of the national motorway (Fig. 1). Then, we followed the grey). same sampling design for each remnant as in the road slopes (see Concostrina-Zubiri et al., 2018, for more details of the study system). We focused only on lichen biocrusts because ing the natural recovery of biocrusts. Beyond road restora- they are the most conspicuous, rich and common compo- tion, this information is critical to the progress of current re- nents of Mediterranean biocrusts (Concostrina-Zubiri et al., search on artificial recovery of biocrusts, a growing body of 2014, 2018) and allowed a relatively easy identification in work in the last years. the field. Other typical biocrust organisms such as cyanobacteria and mosses were not considered in this study, although 2 Material and methods they were present in the road slopes but marginally or not visually apparent (personal observation). Lichen biocrusts 2.1 Study area were identified to the species level when possible, following Nimis and Martellos (2016) and Prieto et al. (2010a, b) This study was conducted in the road slopes and surround- for Placidium. We collected lichen specimens of species that ing natural habitat remnants of a typical Mediterranean gyp- could not be identified to the species level in the field for sum shrubland in central Spain. The study area comprised identification in the laboratory. We identified a total of 18 two adjacent rectangles, each 3 km wide, along both sides taxa: 15 lichens and three lichen groups identified in the of a 13 km segment of the A3 national highway (Fig. 1) field as Enchylium complex (dominated by E. tenax (Sw.) ◦ 0 00 ◦ 0 00 between Fuentidueña del Tajo (40 7 7.96 N, 3 9 41.96 W, Gray, E. coccophorum (Tuck.) Otálora, P. M. Jørg. & Wedin ◦ 0 00 ◦ 0 00 570 m a.s.l.) and Belinchón (40 2 52.95 N, 3 3 29.68 W, and Blennothallia crispa (Hudson) Otálora, P. M. Jørg. & 761 m a.s.l.). The highway goes through an extensive land- Wedin), Gyalolechia complex (including G. fulgens (Sw.) scape of cereal croplands where small patches of natural Søchting, Frödén & Arup and G. subbracteata (Nyl.) Søcht- vegetation remain. The vegetation in these natural patches ing, Frödén & Arup) and Placidium complex (dominated is characterized by an open shrubland dominated by gypso- by P. squamulosum (Ach.) Breuss, P. pilosellum (Breuss) phytes such as Thymus lacaitae Pau, Lepidium subulatum L. Breuss and Clavascidium lacinulatum (Ach.) M. Prieto). and Helianthemum squamatum (L.) Dum. Cours. accompa- Lichen biocrusts are referred to hereafter as “biocrusts” and nied by perennial grasses such as Stipa tenacissima L. and species and species complexes are referred to as “species” Koeleria castellana Boiss. & Reut., among others. Soils are for simplicity. Nomenclature follows Hladun and Llimona predominantly Typic Gypsiorthid (> 70 % gypsum soil con- (2002–2007) and Prieto et al. (2010a, b), except Clavascid- tent). The climate in the area is Mediterranean semiarid with ium lacinulatum (Ach.) M. Prieto, which follows Prieto et a mean annual precipitation of 443 mm and mean annual al. (2012), Enchylium spp., which follow Otálora et al. (2014) ◦ temperature of 14 C, with a very intense summer drought and Gyalolechia spp., which follow Arup et al. (2013). (Ninyerola et al., 2005). 2.3 Characterization of biocrust communities 2.2 Biocrust sampling and identification We characterized biocrust communities by measuring total We selected 17 road slopes along the 13 km highway stretch cover (i.e., mean percentage cover), species richness (i.e., and differentiated two types because of big differences in mean number of biocrust species s.l.), species composition www.web-ecol.net/19/39/2019/ Web Ecol., 19, 39–51, 2019 42 L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes and functional structure at each plot. To assess functional 2.4 Characterization of road slope topography, soil structure, we first classified each species considering differ- properties and vascular plant communities ent ecological features, i.e., eutrophication tolerance, gypsum tolerance and thallus continuity (Table 2). The eutroph- In order to partial out the effect of topography, soil variabil- ication tolerance of biocrusts was classified into two cate- ity and vascular plants in each plot (on both road slopes and gories, following Nimis and Martellos (2017): (1) species natural remnants), we first measured the slope, aspect and not resistant to eutrophication or resistant to very weak eu- latitude in the center of the plot for calculating the heat load trophication (low); (2) species resistant to weak eutrophica- index (McCune and Keon, 2002). The heat load index can tion (medium). The gypsum tolerance of each species was be regarded as a measure of potential solar radiation sym- classified into three categories following Concostrina-Zubiri metrical about the north–south axis and is expected to influ- et al. (2014) and field observations: (1) species that are rarely ence biocrust establishment and development via soil tem- found on gypsum soils (low), (2) species that are less abun- perature and moisture modification, among others (Bowker dant on gypsum soils compared to other substrates (medium), et al., 2016). Then, soil properties were characterized by (3) species generally growing on gypsum soils only (high). collecting one soil sample (5 cm diameter and 10 cm deep) The thallus continuity was classified into three classes fol- under dominant perennial plants (“plant microsite”) in each lowing Nimis and Martellos (2017) and field observations: plot. Also, another sample was collected from areas devoid (1) thallus consisting of disperse squamules or with a fru- of plant cover (“interspace microsite”), which are typically ticose morphology (discontinuous), (2) thallus with more covered with biocrusts. These samples were collected in Au- or less contiguous squamules/lobes or with a foliose mor- gust when the soil was dry. Samples were analyzed for total phology (semicontinuous) and 3) thallus with a crustose– organic C (OC), total N, total P, K, pH, electrical conductiv- placodioid–leprose morphology (continuous). Complexes in- ity (EC), and two soil enzyme activities related to C dynam- cluding several species (i.e., Enchylium, Gyalolechia and ics (β-glucosidase) and P (acid phosphatase) cycles. Labo- Placidium complexes s.l.) were classified as the common cat- ratory techniques used to determine each soil parameter are egory for the species constituting the complexes (i.e., all or explained in Appendix S1. Soil stoniness was also estimated most of the species in the complex shared the same functional as the percentage cover of rock or rock fragments > 10 cm in category; as for Enchylium and Gyalolechia complexes) or as each plot. Finally, the percentage cover of perennial plants the category for the most abundant species in gypsum soils (plant cover, hereafter) in each plot was sampled in a parallel (following Concostrina-Zubiri et al., 2014, and field observa- study (Arenas et al., 2017a) by visual estimation. Taking into tions, as for Placidium complex). Secondly, we evaluated the account the plant cover and the bare ground surface of each functional structure of biocrust communities at each plot cal- plot, we calculated a weighted averaged mean value per soil culating the eutrophication tolerance index, the gypsum tol- variable considering the samples collected at the plant and erance index and the thallus continuity index by means of the interspace microsites. Average values (± SE) for topography community-weighted mean (CWM) for each ecological fea- variables, soil properties and plant cover at each environmen- ture. CWMs were adapted for each multinomial functional tal scenario are shown in Table 3. variable. For instance, we calculated eutrophication tolerance (ET) of each plot by using the following index (1):

n n 2.5 Data analyses X X ET = ET ∗Cover / Cover , (1) sp sp sp To test for differences in total biocrust cover, species richness sp=1 sp=1 and CWM for the three functional indices (i.e., eutrophica- where ETsp is the eutrophication tolerance of each species tion tolerance, gypsum tolerance and thallus continuity) be- (low, medium), Coversp is the estimated cover of each species tween environmental scenarios (i.e., embankments, roadcuts and n is the species richness in each plot. ET can range be- and remnants or roadcuts vs. remnants), we conducted non- tween 1 (all species of a plot show low tolerance) and 2 (all parametric Kruskal–Wallis tests and, when signiﬁcant differ- species show medium tolerance). The CWM for gypsum tol- ences were found, we used the Conover–Iman test for post erance (GT) or thallus continuity (TC) was estimated in the hoc analyses. For total biocrust cover and species richness, same way as ET, replacing ETsp by GTsp or TCsp, with both we included all plots (i.e., eight embankments, nine roadcuts presenting three levels: low (value = 1), medium (value = 2) and 50 natural remnants). However, to calculate the CWM and high (value = 3) for GTsp and discontinuous (value = 1), for the three functional indices, we only included plots where semicontinuous (value = 2) and continuous (value = 3) for lichen biocrust communities and more than one species were TCsp. Therefore, GT and TC can range between 1 and 3. present (McCune and Grace, 2002). Therefore, we limited the analysis to six roadcuts and the 50 natural remnants, while embankments were excluded (i.e., only one species was found in this scenario type). Analyses were performed using the “kruskal()” function in the agricolae R package (de

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Table 1. Biocrust species codes and frequency as number and percentage of plots at each scenario. For roadcuts and embankments, two percentages are presented: the ﬁrst is relative to the total number of plots for each scenario, and the second is relative only to the number of plots where biocrusts were present, in brackets.

Species Code Embankments Roadcuts Natural remnants No % No % No % Acarospora nodulosa var. reagens AcNo 0 0 0 0 12 0.24 Acarospora placodiiformis AcPla 0 0 0 0 7 0.14 Buellia zoharyi BuZo 0 0 0 0 2 0.04 Cetraria aculeata CeAc 0 0 0 0 4 0.08 Cladonia convoluta ClaCo 0 0 2 0.22 (0.33) 30 0.60 Diplotomma alboatrum DiAl 0 0 0 0 14 0.28 Diploschistes diacapsis DiDi 0 0 1 0.11 (0.16) 34 0.72 Enchylium complex EnCo 5 0.62 (1) 6 0.67 (1) 49 0.98 Gyalolechia complex GyCo 0 0 6 0.67 (1) 48 0.96 Leproloma sp. LeSp 0 0 1 0.11 (0.16) 12 0.24 Placidium complex PlaCo 0 0 4 0.44 (0.67) 47 0.94 Placynthium nigrum PlaNi 0 0 2 0.22 (0.33) 18 0.36 Psora decipiens PsoDe 0 0 0 0 44 0.88 Psora globifera PsoGl 0 0 0 0 5 0.10 Psora savizcii PsoSa 0 0 0 0 19 0.38 Squamarina cartilaginea SqCa 0 0 0 0 7 0.14 Squamarina lentigera SqLe 0 0 0 0 38 0.76 Toninia sedifolia ToSe 0 0 2 0.22 (0.33) 45 0.90

Table 2. Biocrust species and functional trait categories. See Ta- RDA is a linear ordination method, like principal component ble 1 for species codes. analysis, where the response variables (i.e., species composition) are modeled as a function of explanatory or con- Eutrophication Gypsum Thallus straining variables (i.e., environmental variables; Zuur et al., Code tolerance tolerance continuity 2007). There are as many RDA axes (“constrained axes”) as AcNo Low High Continuous there are continuous explanatory variables or (n−1) levels in AcPla Low High Continuous nominal explanatory variables. Once all possible RDA axes BuZo Low High Semicontinuous have been calculated, a series of principal component anal- CeAc Low Low Discontinuous ysis (PCA) axes are calculated to maximize the explanation ClaCo Low Medium Semicontinuous of the remaining variation in the data (“unconstrained axes”). DiDi Medium Medium Continuous In our case, we limited the RDA to roadcuts and natural rem- DiEp Medium High Continuous nant scenarios where biocrusts and more than one species EnCo Medium Medium Semicontinuous were present (6 and 50 plots, respectively), as for CWM. This GyCo Medium Medium Semicontinuous means we only used a nominal variable with two levels and, LeSp Low High Continuous thus, only one RDA axis was calculated, followed by PCA PlaCo Medium Medium Semicontinuous PlaNi Medium Low Continuous axes. Because Euclidean distance (used in RDA) is inappro- PsoDe Medium Medium Semicontinuous priate for raw species abundance data involving many zero PsoGl Low High Semicontinuous values, the species abundance matrix was transformed us- PsoSa Low Medium Semicontinuous ing the Hellinger standardization (Legendre and Gallagher, SqCa Medium Medium Continuous 2001). RDA was performed using the “rda()” function in SqLe Low Medium Continuous the vegan R package (Oksanen et al., 2018). Then, to ex- ToSe Medium Medium Semicontinuous amine if topography, soil properties and plant cover influenced the composition of biocrust communities, the afore- mentioned groups of variables were fitted to the RDA, separately, and their significance was tested using the “envfit()” Mendiburu, 2017). The Bonferroni correction was used to function in the vegan R package (Oksanen et al., 2018). Fi- adjust p values for multiple comparisons. nally, three partial RDAs were conducted to determine the The relative importance of the type of environmental sce- relative importance of the type of environmental scenario af- nario on biocrust composition (i.e., species abundance per ter removing the effect of topography, soil and plant cover, site matrix) was evaluated using redundancy analysis (RDA). www.web-ecol.net/19/39/2019/ Web Ecol., 19, 39–51, 2019 44 L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes

Table 3. Average values (± SE) for topography, soil properties and plant cover at each scenario. For each variable the signiﬁcance value of the corresponding Kruskal–Wallis test is given. Different letters indicate signiﬁcant differences (Conover–Iman test for post hoc comparisons, p < 0.05).

Variable p Embankments Roadcuts Natural remnants Topography Slope (◦) 0.000 30.75 ± 3.72 a 42.83 ± 2.41 a 7.52 ± 0.45 b Heat load 0.460 0.85 ± 0.08 a 0.69 ± 0.10 a 0.93 ± 0.01 a Soil property β-glucosidase (µmol PnP g−1 h−1) 0.004 3.23 ± 0.55 a 1.56 ± 0.32 ab 1.35 ± 0.10 b Acid phosphatase (µmol PnP g−1 h−11) 0.167 0.73 ± 0.16 a 0.57 ± 0.07 a 0.51 ± 0.04 a N (%) 0.000 2.06 ± 0.49 ab 1.26 ± 0.21 b 2.69 ± 0.15 a P (mg kg−1) 0.501 0.51 ± 0.09 a 0.38 ± 0.09 a 0.41 ± 0.03 a K (ppm) 0.006 0.12 ± 0.03 ab 0.13 ± 0.04 a 0.05 ± 0.00 b OC (%) 0.000 1.58 ± 0.36 ab 0.83 ± 0.15 b 2.08 ± 0.11 a pH 0.006 8.00 ± 0.06 a 7.77 ± 0.06 ab 7.75 ± 0.03 b EC (dS m−1) 0.002 0.72 ± 0.29 b 1.89 ± 0.08 a 1.64 ± 0.08 a Stoniness (%) 0.000 8.25 ± 3.22 a 26.44 ± 10.86 a 2.30 ± 0.91 b Plant cover (%) 0.540 38.75 ± 7.19 a 29.00 ± 6.13 a 32.24 ± 2.02 a separately. The partial RDAs were carried out using the “rda” function in the vegan R package. The signiﬁcance (Type III ANOVA) of the RDA and the partial RDAs was analyzed using permutation tests with 999 randomizations using the “anova.cca()” function in the vegan R package (Oksanen et al., 2018). All statistical analyses were performed using the R software, version 3.5.1 (http://cran.r-project.org/, last access: 1 December 2018).

3 Results Figure 2. Average values (± SE) for biocrust richness and cover. Rs-Em: embankments; Rs-Rc: roadcuts; Nr: natural remnants. For Biocrusts were naturally present in about two-thirds of both each graph the significance value of the Kruskal–Wallis test is the embankments (five out of eight) and roadcuts (six out of given. Different letters indicate significant differences (Conover– nine, Table 1). We found a total of eight biocrust species in Iman test for post hoc comparisons, p < 0.05) among environmen- road slopes; all of them were present in the roadcuts, but only tal scenarios. one (i.e., Enchylium complex) in the embankments (Table 1). The total number of biocrust species in natural areas was 18, Species richness showed a tendency to increase in the fol- including those present in road slopes. We also found one lowing order: embankments < roadcuts < natural remnants. lichen species in natural areas with a very poorly developed However, this variable was statistically similar in the em- thallus that could not be identified, and it was then discarded bankments and roadcuts (mean species richness of 0.6 and for further analysis in this study. The most frequent species in 2.7, respectively), while it increased significantly (up to 8.8) the roadcuts were Enchylium complex and Gyalolechia com- in the natural remnants (Fig. 2a). Similarly, total biocrust plex, both present in six out of nine plots (Table 1). Addi- cover was significantly lower in the embankments and road- tionally, Gyalolechia complex had the highest mean cover in cuts (ca. 1 % and 5 %, respectively) compared to the natural the roadcut scenario (> 1 %). Leproloma sp. had the maxi- remnants (ca. 12 %, Fig. 2b). mum cover recorded in the roadcuts (ca. 10 %), although this The CWM for eutrophication and gypsum tolerance species was present in only one plot at this scenario type (Ta- was similar in roadcuts and natural remnants (p > 0.050, ble 1). Enchylium complex was the only species found in the Fig. 3a, b), while CWM for thallus continuity was signifi- embankments; present in five out of our plots (Table 1) and cantly lower in the roadcuts compared to the natural remnants with a maximum recorded cover of 2 %. (Fig. 3c).

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4.1 Natural recovery of biocrusts on new habitats Although some biocrust components such as cyanobacteria, algae and even mosses are regarded as early colonizers in primary succession (Belnap and Eldridge, 2003), little is known about their potential to colonize and establish in newly created habitats without human assistance. For example, biocrusts (including lichens and mosses) were found to be the dominant life form in the first 100 m after 10 years of glacier retreat in a Canadian deglaciated val- Figure 3. CWMs (± SE) for eutrophication tolerance (“eutroph. ley (Breen and Lévesque, 2008) and widespread communi- tolerance”), gypsum tolerance and thallus continuity. Rs-Rc: road- ties after ∼ 20 years of lava flows (Jackson, 1971). How- cut; Nr: natural remnants. The significance value of the Kruskal– ever, only a couple of studies have assessed biocrust colo- Wallis test is given for each graph. Different letters indicate sig- nization and establishment in human-made habitats. Gypser nificant differences (Conover–Iman test for post hoc comparisons, et al. (2016, 2015) evaluated the structural and functional de- p < 0.05) among environmental scenarios. velopment of biocrusts in post-mining sites in German drylands. These authors found that 13–23 years after mining activity cessation, biocrust communities were able to colo- The RDA of biocrust composition using the type of sce- nize the new substrate, mostly composed of applied tertiary nario as the constraining variable showed a clear separa- and quaternary materials, initially as green algae–moss com- tion of roadcuts towards the positive end of the RDA1 axis munities and developing into rich and extensive moss–lichen from natural remnants. Also, biocrust composition was sig- carpets over years (Gypser et al., 2016, 2015). Also, Gypser nificantly correlated to OC, EC, K, slope, acid phosphatase, et al. (2015) reported an increase in biocrust NDVI (nor- stoniness and heat load (Fig. 4a). The characteristic species malized difference vegetation index) and chlorophyll fluo- with centroids at this positive extreme were Enchylium com- rescence with biocrust developmental state, evidencing the plex, Gyalolechia complex, Leproloma sp. and Placidium functional recovery of these communities in a relatively short complex (results not shown). The type of scenario (road- time (i.e., < 20 years). Similarly, our results showed that cuts vs. natural remnants) significantly explained (F = 3.51, lichen–biocrusts can recover naturally as fast as 20 years p = 0.001) 6.1 % of variance of the RDA (Fig. 4a). How- after habitat creation, i.e., approximate date of road slope ever, after removing the variation given by topography and construction, but only in roadcuts. It is worth noting that al- soil variables, the partial RDAs (Fig. 4b and c) were not sig- though almost half of the species present in natural remnants nificant (F = 0.96, p = 0.440 and F = 0.75, p = 0.630, re- were found in roadcuts, biocrust richness and cover were still spectively). In contrast, the partial RDA after removing the notably lower than in natural remnants. Nevertheless, these variation given by plant cover (Fig. 4d) remained significant results contrast with estimated recovery rates for biocrusts (F = 3.68, p = 0.002) in a machine-cleared road scenario (Lalley and Viles, 2008), which ranged from the multi-decade to the multi-century scale. The great differences in recovery times are likely due 4 Discussion to climate, semiarid vs. hyper-arid in the case of Lalley and Viles (2008), but also to the availability and proximity of Previous work on unassisted revegetation of newly created biocrust inoculum. Actually, when topsoil surface is removed habitats has mostly focused on vascular plants, while the re- at fine scales, e.g., as in scalped patches of less than 1 m2 and covery of biocrust communities has been disregarded. More- immediately surrounded by biocrust material, biocrust recov- over, to our knowledge, this is the first study assessing nat- ery can be as fast as 3 years for cyanobacteria or lower than ural recovery of biocrusts (i.e., unassisted) in road slopes. 5 years for bryophytes (Dojani et al., 2011; Tian et al., 2006). Our results showed that biocrusts can colonize and establish In our study, remnants of natural habitat with well-developed on road slopes, particularly after soil excavation (i.e., as in biocrusts are only a few metres apart from the road slopes, roadcuts). Surprisingly, biocrust communities developed on which may have favored and speeded up the colonization roadcuts were relatively similar in composition to those on process at our study scale (i.e., sufficient biocrust inoculum some natural remnants. Moreover, we found that topogra- availability). phy and fine-scale soil properties were more important than the type of environmental scenario (i.e., roadcuts vs. natural remnants) in determining biocrust species composition. Nev- ertheless, our results suggest that the scenario type may exert some influence on biocrust establishment and development mediated by the vascular plant community configuration associated with each scenario. www.web-ecol.net/19/39/2019/ Web Ecol., 19, 39–51, 2019 46 L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes

embedded and shallower (more stable) at roadcuts, are likely to promote a faster recovery of biocrusts (Belnap and El- dridge, 2003). Conversely, soil structure and age may also explain the presence of only one species, Enchylium complex, in the embankments where less stable, younger and deeper soils formed by applied materials may dominate. Nev- ertheless, Enchylium spp. have been identified as early successional biocrust species (Belnap and Eldridge, 2003) and play an important ecological role in the development of new habitats; the photobiont of these lichens is a cyanobacteria able to fix N, and thus contribute to the improvement of soil properties for the next successional stages. Although we did not characterize soil texture, this soil property has been found to be different in roadcuts and embankments in other road slopes; i.e., roadcuts have coarser soils than embankments (Jimenez et al., 2011). Indeed, soil stoniness in roadcuts tended to be higher than in embankments in our study area. This may have benefited biocrust development because scattered rocks or rock fragments can act as microclimatic shelters (e.g., reducing solar radiation) Figure 4. (a) RDA using the type of scenario as the constraining and as nutrient and biocrust inoculum traps, among others variable. (b, c, d) Partial RDAs using the type of scenario as the (Bowker et al., 2016). Additionally, we found no significant constraining variable, in which the variation attributable to (b) to- differences in topography between embankments and road- pography, (c) soil and (d) plant cover was removed before adjusting cuts, but slope values tended to be higher in the roadcut sce- the model. RDA ordination plots are based on the first and only nario. Steeper slopes and coarser soils, typically found in RDA axis (RDA1) and the following most explanatory PCA axis roadcuts, may explain the generally lower plant cover in this (PCA1). Variables that significantly adjust to the RDA (p < 0.05) scenario type due to the potential difficulty for seeds to estab- are represented by arrows. lish and grow (Bochet and García-Fayos, 2004) or the lower water availability (Jimenez et al., 2011), among other fac- 4.2 Biocrust structure and composition across tors. In turn, vascular plant cover can also be determinant in environmental scenarios are mostly driven by biocrust establishment and development. topography and edaphic factors On the one hand, it is generally accepted that biocrust communities are more abundant and diverse in open ecosys- Our results also showed that biocrust richness and cover were tems with sparse plant cover (Bowker et al., 2016), where notably lower in road slopes than in adjacent natural areas competition for space is lower. On the other hand, the more and that the newly established communities were character- benign microclimate created by plants (e.g., lower solar ra- ized by a lower thallus continuity CWM. Moreover, strik- diation, higher humidity) in drylands can benefit biocrust ing differences between roadcuts and embankments were physiological performance (Hui et al., 2013; Niemi et al., found in terms of species number. Specifically, only one 2002; Singh et al., 2012), although shaded vs. open environ- species, Enchylium complex, was found on embankments, ment preference can be highly species-specific (Concostrina- while roadcuts and natural remnants had a total of 8 and 18 Zubiri et al., 2014; Eldridge et al., 2002). Moreover, multi- species, respectively. Firstly, these results are likely due to ple relationships between biocrusts and soil properties medi- the typically marked structural dissimilarities between em- ated by plants are expected to play a critical role in biocrust bankments and roadcuts (Arenas et al., 2017a; Jimenez et community configuration, even though they have been poorly al., 2011), although the studied soil properties were gener- studied (Zhang et al., 2016). Here, vascular plant cover ally similar in these scenario types. Roadcuts are constructed tended to be higher in the embankments (39 %) than in the by excavation, generating areas of exposed bedrock and bare roadcuts (29 %, Table 3). However, biocrust presence in the soil, while embankments are constructed by heaping and embankments was represented by only one species and it was compacting material and generally applying topsoil treat- exclusively recorded at those sites with plant cover > 30 % ments (Tormo et al., 2009). The artificially created topsoil (results not shown). It is also worth noting that although we in the embankments is likely to limit the number of biocrust only estimated the cover of perennial plants, we are aware species able to colonize and establish in these scenarios be- that annual plants can occupy almost the totality of peren- cause, among the potential edaphic factors responsible for nial plant interspaces in the studied embankments, but not biocrust establishment, soil stability has been widely recog- in the roadcuts (personal observation). This suggests that nized as one of the most relevant. In particular, older soils, in embankments – habitats with less favorable soil condi-

Web Ecol., 19, 39–51, 2019 www.web-ecol.net/19/39/2019/ L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes 47 tions for biocrusts, as mentioned above – perennial and an- in the studied landscape. Nevertheless, our results also sug- nual plant communities out-compete biocrusts by limiting gest that intrinsic characteristics of roadcuts (e.g., soil ex- habitat space availability. This hypothesis is somehow sup- cavation, generally higher slopes), embankments (e.g., soil ported by the fact that the roadcut with the lowest perennial compacting) and natural remnants may exert an indirect ef- plant cover registered a remarkably high cover of biocrusts fect on biocrust composition mediated by the particular plant (ca. 9 %, results not shown). Notwithstanding, our results community structure and composition associated with each suggest that complex biotic–abiotic interactions operate in environmental scenario. our road slopes. Further work should explore the type (positive vs. negative) and magnitude of the effects that both 4.3 New application of biocrusts as restoration tools in perennial and annual plants, biocrusts and soil properties ex- disturbed drylands: recovery of road slope structure ert on each other at very fine scales to better understand the and function natural recovery process of these newly created habitats. Secondly, we expected that biocrust communities at road- The role of biocrusts as restoration tools in drylands has been cuts and natural remnants would show differences in partic- gaining attention during the last 2 decades. Biocrusts are nat- ular ecological features such as gypsum and eutrophication ural, common components of drylands, where high abiotic tolerance and thallus continuity. Surprisingly, communities at stress conditions (i.e., high solar radiation, low water and nu- both types of scenarios were rather similar in terms of gyp- trient inputs) typically combined with disturbance pressure sum and eutrophication tolerance. However, these findings such as grazing (Belnap, 2003) make them one of the most suggest that roadcuts and natural remnants present small dif- important elements for ecosystem structure and function, in ferences in gypsum content and nutrient inputs, or conversely comparison to other systems dominated by vascular plants. that biocrust species can adapt well to varying environmen- Due to their resistance to harsh environmental conditions and tal conditions related to soil and air composition when suffi- low resource requirements, together with their relative ease cient microhabitat is available (i.e., low plant cover). Also, as of handling and interesting functional attributes (e.g., car- we hypothesized, biocrust communities had a slightly lower bon and nitrogen fixation, soil stabilization, soil–water dy- CWM for thallus continuity in the roadcuts, where less rough namic regulation), biocrusts are regarded as key organisms surfaces can be found. Our results are in accordance with the in dryland restoration research. In fact, higher biocrust cover generally accepted scheme for biocrust succession, where or- has been related to increased soil stability on road slopes in ganisms with smaller structural units (e.g., cyanobacteria and Mediterranean grasslands, contributing to the improvement algae) are the first to colonize new surfaces, while more con- of soil condition even more than vascular plants (García- tinuous covers of lichens and mosses are found with time to- Palacios et al., 2011). Therefore, restoration strategies in dry- gether with increased soil microrelief (Belnap and Eldridge, lands should begin to consider biocrusts as relevant commu- 2003; Williams et al., 2012). nities in the ecosystem recovery process, as opposed to the Finally, our results indicate that a particular combination traditional “vascular-plants only” approach. Indeed, it would of topographic factors and soil properties was shared be- be interesting to test if the high vascular plant cover found tween roadcuts and at least some of the adjacent natural rem- in road slopes (ca. 30 %–40 % of mean cover; Arenas et al., nants, leading to a similar species composition. It is well 2017a) is due to the amelioration of the initial soil conditions known that soil properties such as texture, fertility and pH are by lichen and other biocrust types. strong determinants of biocrust distribution patterns at the in- So far, it has been shown that biocrusts – including lichens termediate and local scales (reviewed in Bowker et al., 2016). – can be grown artificially in the greenhouse rapidly (Do- Also, the solar radiation load can shape biocrust communities herty et al., 2015; Bowker and Antoninka, 2016), and un- through direct effects on biocrust organisms (e.g., ultravio- der field conditions as well (Xiao et al., 2015). Moreover, let radiation received) or indirectly by regulating soil mois- a few experiments have successfully applied biocrusts in ture and vascular plant development (Bowker et al., 2016). the field, and continued growing and functioning have been Accordingly, we found that biocrust composition in roadcuts recorded, at least at the short term (Chiquoine et al., 2016; and natural remnants was, at least in part, driven by topog- Condon and Pyke, 2016; Antoninka et al., 2017). Soil prop- raphy, i.e., slope and heat load, and several soil properties erties such as organic matter content, the inoculum type (i.e., related to soil structure, fertility and functioning, i.e., stoni- field-collected vs. greenhouse-cultured), the climatic similar- ness, EC, soil K, OC and acid phosphatase. Indeed, differ- ity of collection and application zone and the initial species ences in biocrust composition between roadcuts and natural composition of biocrusts to be applied are key points for remnants were mainly due to soil and topographic character- the success of biocrust re-establishment (Condon and Pike, istics, but not to the type of environmental scenario per se. 2016; Bowker and Antoninka, 2016; Bowker et al., 2017; This is in agreement with Concostrina-Zubiri et al. (2018), Antoninka et al., 2017). Here, we show that initial soil char- who reported that soil properties and topography explained acteristics, as given by road slope type, are critical to sustain- biocrust diversity better than habitat structure (e.g., connec- ing relatively rich and diverse biocrusts in a road construction tivity) and disturbance history (i.e., changes in habitat area) context: biocrust restoration efforts should be focused on ex- www.web-ecol.net/19/39/2019/ Web Ecol., 19, 39–51, 2019 48 L. Concostrina-Zubiri et al.: Biocrusts on dryland road slopes cavated areas where bare soil and exposed bedrock dominate bial activity and assess biocrust–vascular plant interactions (e.g., roadcuts). over time. This would allow us to better quantify the contri- Recently, the application of biocrust cyanobacteria inocu- bution of biocrusts to ecosystem development in new land- lum (i.e., assisted recovery) has been identified as an effec- scapes created after the construction of linear infrastructures. tive method for biocrust restoration in road slopes, speed- This is particularly relevant in drylands due to their vulnera- ing up the recovery rate of biocrust cover and function to bility to land-use change (Reynolds et al., 2007). periods shorter than 2 years (Chiquoine et al., 2016). For biocrust lichens, thalli translocation with the use of adhesives such as glue or simply water addition has also been Data availability. Data are available from the Zenodo digital proposed as an effective measure to improve biocrust estab- repository: https://doi.org/10.5281/zenodo.3237017 (Concostrina- lishment and assure functional recovery in disturbed gyp- Zubiri et al., 2019). sum soils (Ballesteros et al., 2017). In this context, common and/or relatively abundant species in road slopes such as Enchylium spp. or Gyalolechia spp. can be considered Supplement. The supplement related to this article is available promising species for active biocrust restoration. Indeed, it online at: https://doi.org/10.5194/we-19-39-2019-supplement. would be interesting to collect specimens of these species at sites that will be converted to road slopes before the dis- Author contributions. LCZ, JMA, IM and AE conceived and de- turbance begins, store them and then transplant them after signed the study, LCZ and JMA conducted field work, JMA and construction. Conversely, our findings suggest that natural LCZ analyzed the data, and LCZ, JMA, IM and AE wrote the pa- colonization can also be regarded as an interesting restora- per. tion option; although ecosystem recovery may take longer to occur, it has the advantage of increasing local diversity in these human-made ecosystems as well as being highly cost- Competing interests. The authors declare that they have no con- effective (Prach and Hobbs, 2008). flict of interest.

5 Conclusions Acknowledgements. L. Concostrina-Zubiri and J. M. Arenas were supported by the REMEDINAL-3 (S2013/MAE-2719) of Biocrust restoration offers an interesting opportunity to re- the Comunidad de Madrid, Spain, and by the European Union cover soil structure and function in disturbed drylands and (Gypworld H2020-MSCA-RISE-2017-777803). We thank the two provides an alternative to traditional restoration techniques anonymous reviewers for their comments, which improved an ear- using vascular vegetation (Bowker, 2007). This study evi- lier version of the paper. dences that lichen biocrusts can establish naturally on newly created habitats without any human assistance. We also show Financial support. This research has been supported by the that topography and edaphic factors related to soil physi- R&D Department of Obrascon Huarte Lain, S.A. (OHL), the cal characteristics (structure of roadcuts vs. embankments, Spanish Ministry of Economy and Competitiveness (grant nos. stoniness), composition and functioning (as indicated by ECONECT CDTI IDI-20120317 and ROOTS-CGL2015-66809-P), OC, K, EC and acid phosphatase values) play an important and the Madrid regional government (grant nos. REMEDINAL-2 role in the configuration of new biocrust communities on S-2009/AMB-1783 and REMEDINAL-3 S2013/MAE-2719). timescales of < 20 years. Actually, we found that roadcuts and natural remnants with similar topography and soil properties shared similar biocrust species composition. Our find- Review statement. This paper was edited by Daniel Montesinos ings thus suggest that given a certain combination of initial and reviewed by two anonymous referees. soil properties and topographic factors, biocrusts will likely develop successfully with no further actions needed. Con- versely, our results provide a number of promising species for active biocrust restoration in Mediterranean road slopes, References since they are common (e.g., Enchylium spp.) or can achieve relatively high cover (e.g., Gyalolechia spp.) in such envi- Antoninka, A., Bowker, M. A., Chuckran, P., Barger, N. N., Reed, S., and Belnap, J.: Maximizing establishment ronmental scenarios. Previous work has pointed out the role and survivorship of field-collected and greenhouse-cultivated of biocrusts in promoting soil stability in road slopes of biocrusts in a semi-cold desert, Plant Soil, 429, 1–13, Mediterranean grasslands (García-Palacios et al., 2011). Fu- https://doi.org/10.1007/s11104-017-3300-3 2017. ture research should evaluate the effect of lichens and other Arenas, J. M., Escudero, A., Mola, I., and Casado, M. A.: Road- biocrust groups (e.g., cyanobacteria) on the improvement of sides: an opportunity for biodiversity conservation, Appl. Veg. topsoil properties such as texture, nutrient content and micro- Sci., 20, 527–537, https://doi.org/10.1111/avsc.12328, 2017a.

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